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Endophytic, saprobic, and pathogenic fungi have evolved elaborate strategies to obtain nutrients from plants. Among the diverse plant-fungi interactions, the most crucial event is the attachment and penetration of the plant surface. Appressoria, specialized infection structures, have been evolved to facilitate this purpose. In this review, we describe the diversity of these appressoria and classify them into two main groups: single-celled appressoria (proto-appressoria, hyaline appressoria, melanized (dark) appressoria) and compound appressoria. The ultrastructure of appressoria, their initiation, their formation, and their function in fungi are discussed. We reviewed the molecular mechanisms regulating the formation and function of appressoria, their strategies to evade host defenses, and the related genomics and transcriptomics. The current review provides a foundation for comprehensive studies regarding their evolution and diversity in different fungal groups.

Salmonella Typhi, the causative agent of typhoid fever, a life-threatening systemic infection, is a human restricted bacterial pathogen. A fundamental aspect of S. Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic S. Typhi infection, the mechanisms that macrophages use to control the growth of S. Typhi and the role of these mechanisms in the bacterium’s adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of S. Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescently-labeled S. Typhi, cells defective in genes important for controlling S. Typhi growth (and therefore unable to kill S. Typhi) are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells.

Over the past decade, the scientific committee has called for broadening our horizons in understanding host–microbe interactions and infectious disease progression. Owing to the fact that the human gut harbors trillions of microbes that exhibit various roles including the production of vitamins, absorption of nutrients, pathogen displacement, and development of the host immune system, particular attention has been given to the use of germ-free (GF) animal models in unraveling the effect of the gut microbiota on the physiology and pathophysiology of the host. In this review, we discuss common methods used to generate GF fruit fly, zebrafish, and mice model systems and highlight the use of these GF model organisms in addressing the role of gut-microbiota in gut-related disorders (metabolic diseases, inflammatory bowel disease, and cancer), and in activating host defense mechanisms and amending pathogenic virulence.

Summary Interferon-inducible guanylate-binding proteins (GBPs) promote cell-intrinsic defense through host cell death. GBPs target pathogens and pathogen-containing vacuoles and promote membrane disruption for release of microbial molecules that activate inflammasomes. GBP1 mediates pyroptosis or atypical apoptosis of Salmonella Typhimurium (STm)- or Toxoplasma gondii (Tg)- infected human macrophages, respectively. The pathogen-proximal detection-mechanisms of GBP1 remain poorly understood, as humans lack functional immunity-related GTPases (IRGs) that assist murine Gbps. Here, we establish that GBP1 promotes the lysis of Tg-containing vacuoles and parasite plasma membranes, releasing Tg-DNA. In contrast, we show GBP1 targets cytosolic STm and recruits caspase-4 to the bacterial surface for its activation by lipopolysaccharide (LPS), but does not contribute to bacterial vacuole escape. Caspase-1 cleaves and inactivates GBP1, and a cleavage-deficient GBP1D192E mutant increases caspase-4-driven pyroptosis due to the absence of feedback inhibition. Our studies elucidate microbe-specific roles of GBP1 in infection detection and its triggering of the assembly of divergent caspase signaling platforms., Graphical Abstract, Highlights • Development of two microscopy assays for microbe/microbe-containing vacuole lysis • Human GBP1 is essential for the lysis of Toxoplasma gondii vacuoles and parasites • Caspase-4 recruitment, but not cytosolic escape of Salmonella, is GBP1 dependent • Caspase-1 cleaves and inactivates GBP1 and suppresses caspase-4-driven pyroptosis, Fisch et al. find that GBP1 targets Toxoplasma vacuolar and parasite membranes for disruption of both membranes. In contrast, appearance of cytosolic Salmonella is GBP1 independent, but caspase-4 recruitment to bacteria and activation is GBP1 dependent. In a negative feedback loop, caspase-1 cleaves GBP1 and suppresses caspase-4-driven pyroptosis during Salmonella infection.

BackgroundSaliva contains a very complex mixture of proteins for defense against microbiological pathogens and for oral food perception. Howler monkeys are Neotropical primates that can consume a mostly leaf diet. They are well known to thrive in highly disturbed habitats where they may cope with a diversity of dietary challenges and infection risks. We aimed to describe the salivary proteome of howlers to contribute to better understanding of their physiology.MethodsWe analyzed the salivary proteins of wild black howler monkeys (Alouatta pigra), by SDS-PAGE-1-D and Nano LC-MS/MS and categorized them by their function involved in host defense and oral food perception.ResultsOur proteomic analysis identified 156 proteins in howler saliva including a number of host defense peptides that are the first line of defense in mammals, such as defensin, cathelicidin, dermcidin, and lactotransferrin, and proteins with anti-bacterial, anti-fungal, and anti-viral capacity, such as IgA, IgG, IgM, BPI, salivary heat shock 70 kDa protein, beta-2-microbulin, and protein S-100. We also identified key proteins necessary for taste perception, including salivary carbonic anhydrase VI, cystatin D, IgA, and fatty acid-binding protein. Proteins to detect astringent foods were identifying, including four members of cystatins (A, B, C and D), lactoperoxidase, and histidine-rich proteins. No chitinase and amylase were identified as would be expected because howlers do not eat insects and little starch. These findings provide basic information to future studies in oral biology, ingestive physiology, and physiological ecology of mammals and non-human primates.

The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor kappa B (NF-kappa B) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response., Author summary The ways that animals combat infection involve complex molecular pathways that are triggered upon microbial challenge. While a great deal is known about which pathways are key to a successful defence response, far less is known about exactly what elements of that response are critical to combat a given infection. Using the fruit fly–a genetic workhorse of Biology–we recently showed that a class of host-encoded antibiotics called “antimicrobial peptides” are essential for defence against bacterial infection, but do not contribute as strongly to defence against fungi. However a number of fly immune peptides remain uncharacterized, possibly explaining this gap in our understanding of the fly antifungal defence. Here we describe a novel antifungal peptide gene of fruit flies, and show that it is a major contributor to the fly antifungal defence response. We also found that this gene seems to regulate a behaviour that flies perform after infection, paralleling exciting recent findings that these genes are involved in neurological processes. Collectively, we clarify a key part of the fly antifungal defence, and contribute an important piece to help explain the logical organization of the immune defence against microbial infection.

The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system., Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role. IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

The blue mussel Mytilus edulis is an intensely studied bivalve in biomonitoring programs worldwide. The lack of detailed descriptions of hemolymph-withdrawal protocols, particularly with regard to the place from where hemolymph could be perfused from, raises questions regarding the exact composition of aspirated hemolymph and does not exclude the possibility of contamination with other body-fluids. This study demonstrates the use of high resolution X-ray computed tomography and histology combined with 3D-reconstruction using AMIRA-software to visualize some important vascular-related anatomic structures of Mytilus edulis. Based on these images, different hemolymph extraction sites used in bivalve research were visualized and described, leading to new insights into hemolymph collection. Results show that hemolymph withdrawn from the posterior adductor muscle could be extracted from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastro-intestinal artery. Furthermore, 3D-reconstructions indicate that puncturing hemolymph from the pericard, anterior aorta, atria and ventricle in a non-invasive way should be possible. Hemolymph withdrawal from the heart is less straightforward and more prone to contamination from the pallial cavity. This study resulted simultaneously in a detailed description and visualization of the vascular-related anatomy of Mytilus edulis.

Life history strategies often shape biological interactions by specifying the parameters for possible encounters, such as the timing, frequency, or way of exposure to parasites. Consequentially, alterations in life‐history strategies are closely intertwined with such interaction processes. Understanding the connection between life‐history alterations and host–parasite interactions can therefore be important to unveil potential links between adaptation to environmental change and changes in interaction processes. Here, we studied how two different host–parasite interaction processes, oral and hemocoelic exposure to bacteria, affect various life histories of the Glanville fritillary butterfly Melitaea cinxia. We either fed or injected adult butterflies with the bacterium Micrococcus luteus and observed for differences in immune defenses, reproductive life histories, and longevity, compared to control exposures. Our results indicate differences in how female butterflies adapt to the two exposure types. Orally infected females showed a reduction in clutch size and an earlier onset of reproduction, whereas a reduction in egg weight was observed for hemocoelically exposed females. Both exposure types also led to shorter intervals between clutches and a reduced life span. These results indicate a relationship between host–parasite interactions and changes in life‐history strategies. This relationship could cast restrictions on the ability to adapt to new environments and consequentially influence the population dynamics of a species in changing environmental conditions., Here, we report differences in how female butterflies adapt life‐history traits upon two distinct infection modes. Oral exposure resulted in a accelerated decrease of clutch sizes but an increase in clutch number, whereas hemocoelic exposure resulted in a reduction of egg weight.

Contains fulltext : 172589.pdf (Publisher’s version ) (Closed access) Despite the importance of immune variation for the symptoms and outcome of Lyme disease, the factors influencing cytokine production during infection with the causal pathogen Borrelia burgdorferi remain poorly understood. Borrelia infection-induced monocyte- and T cell-derived cytokines were profiled in peripheral blood from two healthy human cohorts of Western Europeans from the Human Functional Genomics Project. Both non-genetic and genetic host factors were found to influence Borrelia-induced cytokine responses. Age strongly impaired IL-22 responses, and genetic studies identified several independent QTLs that impact Borrelia-induced cytokine production. Genetic, transcriptomic, and functional validation studies revealed an important role for HIF-1alpha-mediated glycolysis in the cytokine response to Borrelia. HIF-1alpha pathway activation and increase in glycolysis-derived lactate was confirmed in Lyme disease patients. In conclusion, functional genomics approaches reveal the architecture of cytokine production induced by Borrelia infection of human primary leukocytes and suggest a connection between cellular glucose metabolism and Borrelia-induced cytokine production.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infection in infants, causing significant morbidity and mortality. No vaccine or specific, effective treatment is currently available. A more complete understanding of the key components of effective host response to RSV and novel preventative and therapeutic interventions are urgently required. Cathelicidins are host defense peptides, expressed in the inflamed lung, with key microbicidal and modulatory roles in innate host defense against infection. In this article, we demonstrate that the human cathelicidin LL-37 mediates an antiviral effect on RSV by inducing direct damage to the viral envelope, disrupting viral particles and decreasing virus binding to, and infection of, human epithelial cells in vitro. In addition, exogenously applied LL-37 is protective against RSV-mediated disease in vivo, in a murine model of pulmonary RSV infection, demonstrating maximal efficacy when applied concomitantly with virus. Furthermore, endogenous murine cathelicidin, induced by infection, has a fundamental role in protection against disease in vivo postinfection with RSV. Finally, higher nasal levels of LL-37 are associated with protection in a healthy human adult RSV infection model. These data lead us to propose that cathelicidins are a key, nonredundant component of host defense against pulmonary infection with RSV, functioning as a first point of contact antiviral shield and having additional later-phase roles in minimizing the severity of disease outcome. Consequently, cathelicidins represent an inducible target for preventative strategies against RSV infection and may inform the design of novel therapeutic analogs for use in established infection.

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum.

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKβ and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKβ and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKβ to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity. Goto et al. show that the kinase dIKKβ and the NF-κB factor Relish control replication of picorna-like viruses in flies through induction of antiviral genes, including a homolog of STING. A STING-IKKβ-NF-κB cassette participates in resistance to picorna-like viruses, pointing to an evolutionarily ancient role of STING in antiviral immunity.

Myeloid C-type lectin receptors (CLRs) are important sensors of self and non-self that work in concert with other pattern recognition receptors (PRRs). CLRs have been previously classified based on their signaling motifs as activating or inhibitory receptors. However, specific features of the ligand binding process may result in distinct signaling through a single motif, resulting in the triggering of non-canonical pathways. In addition, CLR ligands are frequently exposed in complex structures that simultaneously bind different CLRs and other PRRs, which lead to integration of heterologous signaling among diverse receptors. Herein, we will review how sensing by myeloid CLRs and crosstalk with heterologous receptors is modulated by many factors affecting their signaling and resulting in differential outcomes for immunity and inflammation. Finding common features among those flexible responses initiated by diverse CLR-ligand partners will help to harness CLR function in immunity and inflammation. CF is supported by AECC Foundation as recipient of an ``Ayuda Fundacion Cientifica AECC a personal investigador en cancer.´´ SI is funded by grant SAF2015-74561-JIN from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Fund for Regional Development (FEDER). PS-L is funded by grant BES-2015-072699 (´´Ayudas para contratos predoctorales para la formacion de doctores 2015´´) from MINECO. MM-L received a FPU fellowship (AP2010-5935) from the Spanish Ministry of Education. Work in the DS laboratory is funded by the CNIC and grant SAF2016-79040-R from MINECO and FEDER; B2017/BMD-3733 Immunothercan-CM from Comunidad de Madrid; RD16/0015/0018-REEM from FIS-Instituto de Salud Carlos III, MINECO, and FEDER; Foundation Acteria; Constantes y Vitales prize (Atresmedia); Foundation La Marato de TV3 (201723); the European Commission (635122-PROCROP H2020); and the European Research Council (ERC-Consolidator Grant 725091). The CNIC is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). The authors have no conflicting financial interests. Sí

Shigella is a leading cause of dysentery worldwide, responsible for up to 165 million cases of shigellosis each year. Shigella is also recognised as an exceptional model pathogen to study key issues in cell biology and innate immunity. Several infection models have been useful to explore Shigella biology; however, we still lack information regarding the events taking place during the Shigella infection process in vivo. Here, we discuss a selection of mechanistic insights recently gained from studying Shigella infection of zebrafish (Danio rerio), with a focus on cytoskeleton rearrangements and cellular immunity. We also discuss how infection of zebrafish can be used to investigate new concepts underlying infection control, including emergency granulopoiesis and the use of predatory bacteria to combat antimicrobial resistance. Collectively, these insights illustrate how Shigella infection of zebrafish can provide fundamental advances in our understanding of bacterial pathogenesis and vertebrate host defence. This information should also provide vital clues for the discovery of new therapeutic strategies against infectious disease in humans., Summary: Here, we review how studying Shigella infection of zebrafish has illuminated novel research avenues in both infection and cell biology.

Because excessive or inadequate responses can be detrimental, immune responses to infection require appropriate regulation. Networks of signaling pathways establish versatility of immune responses. Drosophila melanogaster is a powerful model organism for dissecting conserved innate immune responses to infection. For example, the Toll pathway, which promotes activation of NF-κB transcription factors Dorsal/Dorsal-related immune factor (Dif), was first identified in Drosophila. Together with the IMD pathway, acting upstream of NF-κB transcription factor Relish, these pathways constitute a central immune signaling network. Inputs in these pathways contribute to specific and appropriate responses to microbial insults. Relish activity during infection is modulated by Ca2+-dependent serine/threonine phosphatase calcineurin, an important target of immunosuppressants in transplantation biology. Only one of the three Drosophila calcineurin isoforms, calcineurin A1, acts on Relish during infection. However, it is not known whether there is a role for calcineurin in Dorsal/Dif immune signaling. In this article, we demonstrate involvement of specific calcineurin isoforms, protein phosphatase at 14D (Pp2B-14D)/calcineurin A at 14F (CanA-14F), in Toll-mediated immune signaling. These isoforms do not affect IMD signaling. In cell culture, pharmacological inhibition of calcineurin or RNA interference against homologous calcineurin isoforms Pp2B-14D/CanA-14F, but not against isoform calcineurin A1, decreased Toll-dependent Dorsal/Dif activity. A Pp2B-14D gain-of-function transgene promoted Dorsal nuclear translocation and Dorsal/Dif activity. In vivo, Pp2B-14D/CanA-14F RNA interference attenuated the Dorsal/Dif-dependent response to infection without affecting the Relish-dependent response. Altogether, these data identify a novel input, calcineurin, in Toll immune signaling and demonstrate involvement of specific calcineurin isoforms in Drosophila NF-κB signaling.

Macrophages provide front line defense against infections. The study of macrophage–microbe interplay is thus crucial for understanding pathogenesis and infection control. Zebrafish (Danio rerio) larvae provide a unique platform to study macrophage–microbe interactions in vivo, from the level of the single cell to the whole organism. Studies using zebrafish allow non-invasive, real-time visualization of macrophage recruitment and phagocytosis. Furthermore, the chemical and genetic tractability of zebrafish has been central to decipher the complex role of macrophages during infection. Here, we discuss the latest developments using zebrafish models of bacterial and fungal infection. We also review novel aspects of macrophage biology revealed by zebrafish, which can potentiate development of new therapeutic strategies for humans.

International audience; The Mexican burrowing toad Rhinophrynus dorsalis is the sole extant representative of the Rhinophrynidae. United in the superfamily Pipoidea, the Rhinophrynidae is considered to be the sister-group to the extant Pipidae which comprises Hymenochirus, Pipa, Pseudhymenochirus and Xenopus. Cationic, α-helical host-defense peptides of the type found in Hymenochirus, Pseudhymenochirus, and Xenopus species (hymenochirins, pseudhymenochirins, magainins, and peptides related to PGLa, XPF, and CPF) were not detected in norepinephrine-stimulated skin secretions of R. dorsalis. Skin secretions of representatives of the genus Pipa also do not contain cationic α-helical host-defense peptides which suggest, as the most parsimonious hypothesis, that the ability to produce such peptides by frogs within the Pipidae family arose in the common ancestor of (Hymenochirus+Pseudhymenochirus)+Xenopus after divergence from the line of evolution leading to extant Pipa species. Peptidomic analysis of the R. dorsalis secretions led to the isolation of rhinophrynin-27, a proline-arginine-rich peptide with the primary structure ELRLPEIARPVPEVLPARLPLPALPRN, together with rhinophrynin-33 containing the C-terminal extension KMAKNQ. Rhinophrynin-27 shows limited structural similarity to the porcine multifunctional peptide PR-39 but it lacks antimicrobial and cytotoxic activities. Like PR-39, the peptide adopts a poly-l-proline helix but some changes in the circular dichroism spectrum were observed in the presence of anionic sodium dodecylsulfate micelles consistent with the stabilization of turn structures.

Tumor necrosis factor-alpha (TNF-α) blockade is an effective treatment for rheumatoid arthritis (RA) and other inflammatory diseases, but in patients, it is associated with reduced resistance to the infectious agents Mycobacterium tuberculosis and Listeria monocytogenes, among others. Our goal was to model infection and arthritis in mice and to compare etanercept, a currently used anti-TNF-α inhibitor, to an anti-TNF-α vaccine. We developed a murine surrogate of the TNF-α kinoid and produced an anti-murine TNF-α vaccine (TNFKi) composed of keyhole limpet hemocyanin conjugated to TNF-α, which resulted in anti-TNF-α antibody production in mice. We also used etanercept (a soluble receptor of TNF commonly used to treat RA) as a control of TNF neutralization. In a mouse model of collagen-induced arthritis, TNFKi protected against inflammation similar to etanercept. In a mouse model of acute L. monocytogenes infection, all TNFKi-treated mice showed cleared bacterial infection and survived, whereas etanercept-treated mice showed large liver granulomas and quickly died. Moreover, TNFKi mice infected with the virulent H37Rv M. tuberculosis showed resistance to infection, in contrast with etanercept-treated mice or controls. Depending on the TNF-α blockade strategy, treating arthritis with a TNF-α inhibitor could result in a different profile of infection suceptibility. Our TNFKi vaccine allowed for a better remaining host defense than did etanercept.

The interferon (IFN)-stimulated gene 15 (ISG15) encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15 -/- and ISG15 +/+ bone marrow derived macrophages (BMDM) after vaccinia virus (VACV) infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) were pathways altered in ISG15 -/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production was higher in ISG15 +/+ BMDM than in ISG15 -/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15 -/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15 +/+ macrophages than in ISG15 -/- macrophages concomitant with a decrease in viral titer. Thus, ISG15 -/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent, This research is supported by a grant from Spanish Ministry of Economy and Competitiveness (MINECO) to SG (SAF2014-54623-R), and to JV from MINECO (BIO2015-67580-P) and from the Carlos III Institute of Health-Fondo de Investigaciónn Sanitaria (PRB2, IPT13/0001ÐISCIII-SGEFI/FEDER, ProteoRed)

Made available in DSpace on 2019-09-12T16:53:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2017 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Due to the growing number of multi-resistant Candida spp., adjuvant treatments that may help combat these fungal pathogens are relevant and useful. This study evaluated the immunomodulation and anti-Candida activity of Lactobacillus rhamnosus (LR), Lactobacillus acidophilus and Lactobacillus paracasei suspensions, either single- or multiple-strain, in mouse macrophages (RAW 264.7) and Galleria mellonella (GM). Mouse macrophages were activated by different lactobacilli suspensions and challenged with C albicans (CA). Tumor necrosis factor (TNF)-alpha, interleukin IL-beta, IL-6 and IL-17 production and cell viability were investigated. LR was the best suspension for stimulating all evaluated cytokines and thus was used in subsequent in vivo assays. Two C albicans clinical strains, CA21 and CA60, were then added to the GM assays to further confirm the results. LR suspension was injected into the larvae 24 h before challenging with CA. Survival curve, CFU per larva and hemocytes were counted. In the GM, the LR suspension increased the survival rate and hemocyte counts and decreased the CFU per larva counts for all groups. Lactobacilli suspensions presented strain-dependent immunomodulation; however, single suspensions showed better results. Anti-Candida activity was demonstrated by decreased Candida counts in the GM with the use of LR. (C) 2017 Elsevier Ltd. All rights reserved. [de Oliveira, Felipe Eduardo; Rossoni, Rodnei Dennis; de Barros, Patricia Pimentel; Begnini, Barbara Evelyn; Junqueira, Juliana Campos; Cardoso Jorge, Antonio Olavo; de Oliveira, Luciane Dias] Sao Paulo State Univ Unesp, Dept Biosci & Oral Diag, Inst Sci & Technol, Ave Engn Francisco Jose Longo 777, BR-12245000 Sao Paulo, Brazil [Pereira Leao, Mariella Vieira] Universidade de Taubaté (Unitau), Sch Med, Inst Basic Biosci, Av Tiradentes 500, BR-12030180 Sao Paulo, Brazil

Background Immunometabolism plays a central role in many cardiometabolic diseases. However, a robust map of immune-related gene networks in circulating human cells, their interactions with metabolites, and their genetic control is still lacking. Here, we integrate blood transcriptomic, metabolomic, and genomic profiles from two population-based cohorts (total N = 2168), including a subset of individuals with matched multi-omic data at 7-year follow-up. Results We identify topologically replicable gene networks enriched for diverse immune functions including cytotoxicity, viral response, B cell, platelet, neutrophil, and mast cell/basophil activity. These immune gene modules show complex patterns of association with 158 circulating metabolites, including lipoprotein subclasses, lipids, fatty acids, amino acids, small molecules, and CRP. Genome-wide scans for module expression quantitative trait loci (mQTLs) reveal five modules with mQTLs that have both cis and trans effects. The strongest mQTL is in ARHGEF3 (rs1354034) and affects a module enriched for platelet function, independent of platelet counts. Modules of mast cell/basophil and neutrophil function show temporally stable metabolite associations over 7-year follow-up, providing evidence that these modules and their constituent gene products may play central roles in metabolic inflammation. Furthermore, the strongest mQTL in ARHGEF3 also displays clear temporal stability, supporting widespread trans effects at this locus. Conclusions This study provides a detailed map of natural variation at the blood immunometabolic interface and its genetic basis, and may facilitate subsequent studies to explain inter-individual variation in cardiometabolic disease., published version, peerReviewed

Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa.

Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. Infection by S. flexneri has been well-studied in vitro and is a paradigm for bacterial interactions with the host immune system. Recent work has revealed that components of the cytoskeleton have important functions in innate immunity and inflammation control. Septins, highly conserved cytoskeletal proteins, have emerged as key players in innate immunity to bacterial infection, yet septin function in vivo is poorly understood. Here, we use S. flexneri infection of zebrafish (Danio rerio) larvae to study in vivo the role of septins in inflammation and infection control. We found that depletion of Sept15 or Sept7b, zebrafish orthologs of human SEPT7, significantly increased host susceptibility to bacterial infection. Live-cell imaging of Sept15-depleted larvae revealed increasing bacterial burdens and a failure of neutrophils to control infection. Strikingly, Sept15-depleted larvae present significantly increased activity of Caspase-1 and more cell death upon S. flexneri infection. Dampening of the inflammatory response with anakinra, an antagonist of interleukin-1 receptor (IL-1R), counteracts Sept15 deficiency in vivo by protecting zebrafish from hyper-inflammation and S. flexneri infection. These findings highlight a new role for septins in host defence against bacterial infection, and suggest that septin dysfunction may be an underlying factor in cases of hyper-inflammation., Author summary Shigella are human-adapted Escherichia coli and cause bacillary dysentery via inflammatory destruction of the gut epithelium. In this study, we use a zebrafish (Danio rerio) model of Shigella infection to discover new roles for the cytoskeleton in inflammation and infection control. Septins, a poorly understood component of the cytoskeleton, are important in numerous biological processes including cell division and host-pathogen interactions. Here, we show that zebrafish septins can restrict inflammation and Shigella infection in vivo. In the absence of septins, larvae infected with Shigella exhibit increased mortality and bacterial burdens associated with increased Caspase-1 activity and neutrophil death. Pharmacological suppression of Il-1β signaling rescues septin-deficiency in vivo by reducing neutrophil death and preventing larval mortality. These findings reveal a new link between septins and inflammation, and highlight the cytoskeleton as a structural determinant of host defence.

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.

Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses.

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30–40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection. In addition to their role in thrombosis and hemostasis, platelets can also migrate to sites of infection to help trap bacteria and clear the vascular surface.

The protozoan parasite Giardia is a highly prevalent intestinal pathogen with a wide host range. Data obtained in mice, cattle and humans revealed the importance of IL-17A in the development of a protective immune response against Giardia. The aim of this study was to further unravel the protective effector mechanisms triggered by IL-17A following G. muris infection in mice, by an RNA-sequencing approach. C57BL/6 WT and C57BL/6 IL-17RA KO mice were orally infected with G. muris cysts. Three weeks post infection, intestinal tissue samples were collected for RNA-sequencing, with samples from uninfected C57BL/6 WT and C57BL/6 IL-17RA KO animals serving as negative controls. Differential expression analysis showed that G. muris infection evoked the transcriptional upregulation of a wide array of genes, mainly in animals with competent IL-17RA signaling. IL-17RA signaling induced the production of various antimicrobial peptides, such as angiogenin 4 and α- and β-defensins and regulated complement activation through mannose-binding lectin 2. The expression of the receptor that regulates the secretion of IgA into the intestinal lumen, the polymeric immunoglobulin receptor, was also dependent on IL-17RA signaling. Interestingly, the transcriptome data showed for the first time the involvement of the circadian clock in the host response following Giardia infection.

Down syndrome cell adhesion molecule 1 ( Dscam1 ) has wide-reaching and vital neuronal functions although the role it plays in insect and crustacean immunity is less well understood. In this study, we combine different approaches to understand the roles that Dscam1 plays in fitness-related contexts in two model insect species. Contrary to our expectations, we found no short-term modulation of Dscam1 gene expression after haemocoelic or oral bacterial exposure in Tribolium castaneum , or after haemocoelic bacterial exposure in Drosophila melanogaster. Furthermore, RNAi-mediated Dscam1 knockdown and subsequent bacterial exposure did not reduce T. castaneum survival. However, Dscam1 knockdown in larvae resulted in adult locomotion defects, as well as dramatically reduced fecundity in males and females. We suggest that Dscam1 does not always play a straightforward role in immunity, but strongly influences behaviour and fecundity. This study takes a step towards understanding more about the role of this intriguing gene from different phenotypic perspectives.

Although obesity is implicated in numerous health complications leading to increased mortality, the relationship between obesity and outcomes for critically ill patients appears paradoxical. Recent studies have reported better outcomes and lower levels of inflammatory cytokines in obese patients with acute lung injury (ALI)/acute respiratory distress syndrome, suggesting that obesity may ameliorate the effects of this disease. We investigated the effects of obesity in leptin-resistant db/db obese and diet-induced obese mice using an inhaled LPS model of ALI. Obesity-associated effects on neutrophil chemoattractant response were examined in bone marrow neutrophils using chemotaxis and adoptive transfer; neutrophil surface levels of chemokine receptor CXCR2 were determined by flow cytometry. Airspace neutrophilia, capillary leak, and plasma IL-6 were all decreased in obese relative to lean mice in established lung injury (24 h). No difference in airspace inflammatory cytokine levels was found between obese and lean mice in both obesity models during the early phase of neutrophil recruitment (2–6 h), but early airspace neutrophilia was reduced in db/db obese mice. Neutrophils from uninjured obese mice demonstrated diminished chemotaxis to the chemokine keratinocyte cytokine compared with lean control mice, and adoptive transfer of obese mouse neutrophils into injured lean mice revealed a defect in airspace migration of these cells. Possibly contributing to this defect, neutrophil CXCR2 expression was significantly lower in obese db/db mice, and a similar but nonsignificant decrease was seen in diet-induced obese mice. ALI is attenuated in obese mice, and this blunted response is in part attributable to an obesity-associated abnormal neutrophil chemoattractant response.

SummaryPeptidoglycan recognition proteins (PGRPs) are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings support the role of PGRP-LB as a negative regulator of the Imd pathway and brought to light a synergy of PGRP-SCs with PGRP-LB in the systemic response. Flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to innocuous gut infections. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. Analysis of the immune phenotype of flies lacking all catalytic PGRPs and the Imd regulator Pirk reveals that the Imd-mediated immune response is highly constrained by the existence of multiple negative feedbacks.

Staphylococcus aureus is a pathogen that often causes severe nosocomial infections including pneumonia. The present study was designed to examine innate phagocyte mediated immune mechanisms using a previously described murine S. aureus Newman pneumonia model. We found that BALB/c mice represent a more susceptible mouse strain compared to C57BL/6 mice after intranasal S. aureus Newman challenge. Depletion experiments revealed that neutrophils are a crucial determinant for resistance whereas depletion of alveolar macrophages protected mice to some degree from acute pulmonary S. aureus challenge. C57BL/6 mice lacking the subunit gp91phox of the NADPH-oxidase (gp91phox-/- mice) proved to be highly susceptible against the pathogen. In contrast, C57BL/6 inducible nitric oxidase synthase deficient (iNOS-/-) mice did not differ in their clinical outcome after infection. Neither bone marrow macrophages from iNOS-/- nor from gp91phox-/- mice were impaired in controlling intracellular persistence of S. aureus. Our data suggest that neutrophil and NADPH-oxidase mediated mechanisms are essential components in protecting the host against pulmonary S. aureus Newman challenge. On contrary, macrophages as well as NO mediated mechanisms do not seem to play a critical role for resistance in this model.

Adjuvants are essential components of most clinically used vaccines. This is because the majority of nonliving vaccines are relatively poor inducers of adaptive immunity unless effective adjuvants are co-administered. Aluminum salts (alum) have been used as adjuvants with great success for almost a century and have been particularly effective at promoting protective humoral immunity. However, alum is not optimally effective for diseases where cell-mediated immunity is required for protection. Furthermore, adjuvants including oil-in-water emulsions have shown improved efficacy for avian influenza protection suggesting that even for diseases where humoral immunity can confer protection, there is scope for developing improved adjuvants. There have been major developments in antigen discovery over the past decade, which has accelerated the vaccine development process for new indications and this demands a new generation of adjuvants that can drive and specifically direct the desired immune responses. A number of systems are under investigation that combine different types of adjuvants into specific formulations with greater activity. Additionally, targeting of vaccines to specific immune cells shows great promise. In the case of cancer and chronic infectious diseases, it may be difficult to develop effective vaccines without blocking immune regulatory pathways, which impede cell-mediated responses. However, increased understanding of immunology and particularly the innate immune system is informing vaccine adjuvant research and consequently driving the development of novel and specifically directed vaccine adjuvant strategies. In this article we address the importance of adjuvants in vaccine development, the known mode of action of specific adjuvants and recent developments in this important field.

The peritrophic matrix (PM) forms a layer composed of chitin and glycoproteins that lines the insect intestinal lumen. This physical barrier plays a role analogous to that of mucous secretions of the vertebrate digestive tract and is thought to protect the midgut epithelium from abrasive food particles and microbes. Almost nothing is known about PM functions in Drosophila , and its function as an immune barrier has never been addressed by a genetic approach. Here we show that the Drosocrystallin (Dcy) protein, a putative component of the eye lens of Drosophila , contributes to adult PM formation. A loss-of-function mutation in the dcy gene results in a reduction of PM width and an increase of its permeability. Upon bacterial ingestion a higher level of expression of antibacterial peptides was observed in dcy mutants, pointing to an influence of this matrix on bacteria sensing by the Imd immune pathway. Moreover, dcy -deficient flies show an increased susceptibility to oral infections with the entomopathogenic bacteria Pseudomonas entomophila and Serratia marcescens . Dcy mutant flies also succumb faster than wild type upon ingestion of a P. entomophila toxic extract. We show that this lethality is due in part to an increased deleterious action of Monalysin, a pore-forming toxin produced by P. entomophila . Collectively, our analysis of the dcy immune phenotype indicates that the PM plays an important role in Drosophila host defense against enteric pathogens, preventing the damaging action of pore-forming toxins on intestinal cells.

Species of the Sporothrix complex are the etiological agents of sporotrichosis, an important subcutaneous mycosis with several clinical forms and an increasing incidence around the world that affects humans and other mammals. The immunological mechanisms involved in the prevention and control of this mycosis are not entirely understood. Many reports have suggested that cell-mediated immunity has an essential role in the development of the disease, being the primary response controlling it, while only recent data supports that the humoral response is essential for the appropriate control. This mycosis is a challenge for diagnosis since the culture and isolation of the organism are time-consuming and complicated; reasons that have led to the study of fungus antigenic molecules capable of generating a detectable humoral response. The treatment for this disease includes the use of several antifungal drugs like itraconazole, amphotericin B, caspofungin, fluconazole, and the combination between them among others such as the extract of Vismia guianensis.

Wathes DC, Cheng Z, Chowdhury W, Fenwick MA, Fitzpatrick R, Morris DG, Patton J, Murphy JJ. Negative energy balance alters global gene expression and immune responses in the uterus of postpartum dairy cows. Physiol Genomics 39: 1-13, 2009. First published June 30, 2009; doi: 10.1152/physiolgenomics.00064.2009.-Most dairy cows suffer uterine microbial contamination postpartum. Persistent endometritis often develops, associated with reduced fertility. We used a model of differential feeding and milking regimes to produce cows in differing negative energy balance status in early lactation (mild or severe, MNEB or SNEB). Blood hematology was assessed preslaughter at 2 wk postpartum. RNA expression in endometrial samples was compared using bovine Affymetrix arrays. Data were mapped using Ingenuity Pathway Analysis. Circulating concentrations of IGF-I remained lower in the SNEB group, whereas blood nonesterified fatty acid and beta-hydroxybutyrate concentrations were raised. White blood cell count and lymphocyte number were reduced in SNEB cows. Array analysis of endometrial samples identified 274 differentially expressed probes representing 197 recognized genes between the energy balance groups. The main canonical pathways affected related to immunological and inflammatory disease and connective tissue disorders. Inflammatory response genes with major upregulation in SNEB cows included matrix metalloproteinases, chemokines, cytokines, and calgranulins. Expression of several interferon-inducible genes including ISG20, IFIH1, MX1, and MX2 were also significantly increased in the SNEB cows. These results provide evidence that cows in SNEB were still undergoing an active uterine inflammatory response 2 wk postpartum, whereas MNEB cows had more fully recovered from their energy deficit, with their endometrium reaching a more advanced stage of repair. SNEB may therefore prevent cows from mounting an effective immune response to the microbial challenge experienced after calving, prolonging the time required for uterine recovery and compromising subsequent fertility.

Largely viewed as proinflammatory, innate responses combine with adaptive immunity to generate the most effective form of antifungal resistance, and T cells exercise feedback control over diverse effects of inflammation on infection. Some degree of inflammation is required for protection, particularly in mucosal tissues, during the transitional response occurring between the rapid innate and slower adaptive response. However, progressive inflammation worsens disease and ultimately prevents pathogen eradication. IDO, tryptophan catabolites (“kynurenines”), and regulatory T cells help to tame overzealous and exaggerated inflammatory responses. In this context, IL-23 and the Th17 pathway, which down-regulate tryptophan catabolism, may instead favor pathology and serve to accommodate the seemingly paradoxical association of chronic inflammation with fungal persistence. Recent data support a view in which IL-23/IL-17 antagonistic strategies, including the administration of synthetic kynurenines, could represent a new means of harnessing progressive or potentially harmful inflammation.

We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand beta-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) gamma strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by beta-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor beta, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4(+) T cells, IL-1beta, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.

Hot-water extracts of the higher Basidiomycetes Agaricus bisporus (J. Lge) Imbach, A. brasiliensis S. Wasser et al., Coprinus comatus (O.F. Mull.) Pers., Ganoderma lucidum (W. Curt.: Fr.) P. Karst., and Phellinus linteus (Berk. et Curt.) Teng were made, and the resulting polysaccharide mixtures were purified by DEAF-cellulose chromatography and ethanol precipitation. The extracts were noncytopathic. A. bisporus, A. brasiliensis, and G. lucidum strongly generated reactive oxygen species (ROS) in human PBMCs and K 562 cells. C. comatus and Ph. linteus extracts had low ROS-generating capacity. In A. bisporus extract, two different classes of polysaccharides were found. The colorless polysaccharide of MW > 250 kDa caused no intracellular ROS generation; acid hydrolysis followed by HPLC analysis showed it to consist of only glucose residues, thus being a pure glucan. The light brown polyphenol/polysaccharide complex of MW 75-200 kDa was found to strongly generate ROS. ROS generation by medicinal mushrooms could therefore be due to the presence of polyphenols rather than of glucan alone. A. bisporus polysaccharide extract showed a saturation level of ROS generation at 2 mg mL(-1). Generation appeared to continue in the presence of polysaccharide for more than 14 hours. Purified colorless polysaccharides of C. comatus and Ph. linteus showed no ROS generation in K562 cells; G. lucidum and A. brasiliensis polysaccharides could not completely be cleaned of phenolic compounds and remained active. Competitive inhibition of ROS generation by laminarin was not observed for any of the polysaccharide extracts, suggesting that polyphenol/glucan complexes isolated from higher Basidiomycetes mushrooms are able to generate ROS without binding to a dectin receptor.

Summary Fungal infections are major causes of morbidity and mortality, especially in immunocompromised individuals. The innate immune system senses fungal pathogens through Syk-coupled C-type lectin receptors (CLRs), which signal through the conserved immune adaptor Card9. Although Card9 is essential for antifungal defense, the mechanisms that couple CLR-proximal events to Card9 control are not well defined. Here, we identify Vav proteins as key activators of the Card9 pathway. Vav1, Vav2, and Vav3 cooperate downstream of Dectin-1, Dectin-2, and Mincle to engage Card9 for NF-κB control and proinflammatory gene transcription. Although Vav family members show functional redundancy, Vav1/2/3−/− mice phenocopy Card9−/− animals with extreme susceptibility to fungi. In this context, Vav3 is the single most important Vav in mice, and a polymorphism in human VAV3 is associated with susceptibility to candidemia in patients. Our results reveal a molecular mechanism for CLR-mediated Card9 regulation that controls innate immunity to fungal infections., Graphical Abstract, Highlights • Vav proteins control CLR-mediated inflammatory responses • CLR-induced NF-κB activation is regulated by Vav proteins • Vav/Card9 signaling is critical for antifungal host defense, CLR/Card9 signaling is essential for antifungal immunity. Roth et al. have identified the Vav family of proteins as key activators of the Card9/NF-kB pathway, essential for CLR-induced inflammatory responses and host defense against fungi.

Biofilm has been recognized as a well-protected form of living for bacteria, contributing to bacterial pathogenicity, particularly for opportunistic species. Biofilm-associated infections are marked by their persistence. Extensive research has been devoted to the formation and composition of biofilms. The immune response against biofilms remains rather unexplored, but there is the notion that bacteria within a biofilm are protected from host defences. Here we glance at the mechanisms by which neutrophils recognize and face biofilms in implant infections and discuss the implications of this interplay, as well as speculate on its significance.

The Gram-positive bacterium Streptococcus pneumoniae causes life-threatening infections, especially among immunocompromised patients. The host's immune system senses S. pneumoniae via different families of pattern recognition receptors, in particular the Toll-like receptor (TLR) family that promotes immune cell activation. Yet, while single TLRs are dispensable for initiating inflammatory responses against S. pneumoniae, the central TLR adapter protein myeloid differentiation factor 88 (MyD88) is of vital importance, as MyD88-deficient mice succumb rapidly to infection. Since MyD88 is ubiquitously expressed in hematopoietic and non-hematopoietic cells, the extent to which MyD88 signaling is required in different cell types to control S. pneumoniae is unknown. Therefore, we used novel conditional knockin mice to investigate the necessity of MyD88 signaling in distinct lung-resident myeloid and epithelial cells for the initiation of a protective immune response against S. pneumoniae. Here, we show that MyD88 signaling in lysozyme M (LysM)- and CD11c-expressing myeloid cells, as well as in pulmonary epithelial cells, is critical to restore inflammatory cytokine and antimicrobial peptide production, leading to efficient neutrophil recruitment and enhanced bacterial clearance. Overall, we show a novel synergistic requirement of compartment-specific MyD88 signaling in S. pneumoniae immunity.

The enteropathogen Salmonella Typhimurium (S. Typhimurium) is the most commonly non-typhoideal serotype isolated in pig worldwide. Currently, one of the main sources of human infection is by consumption of pork meat. Therefore, prevention and control of salmonellosis in pigs is crucial for minimizing risks to public health. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to explore differences in the response to Salmonella in two segment of the porcine gut (ileum and colon) along a time course of 1, 2, and 6 days post infection (dpi) with S. Typhimurium. A total of 298 proteins were identified in the infected ileum samples of which, 112 displayed significant expression differences due to Salmonella infection. In colon, 184 proteins were detected in the infected samples of which 46 resulted differentially expressed with respect to the controls. The higher number of changes in protein expression was quantified in ileum at 2 dpi. Further biological interpretation of proteomics data using bioinformatics tools demonstrated that the expression changes in colon were found in proteins involved in cell death and survival, tissue morphology or molecular transport at the early stages and tissue regeneration at 6 dpi. In ileum, however, changes in protein expression were mainly related to immunological and infection diseases, inflammatory response or connective tissue disorders at 1 and 2 dpi. iTRAQ has proved to be a proteomic robust approach allowing us to identify ileum as the earliest response focus upon S. Typhimurium in the porcine gut. In addition, new functions involved in the response to bacteria such as eIF2 signaling, free radical scavengers or antimicrobial peptides (AMP) expression have been identified. Finally, the impairment at of the enterohepatic circulation of bile acids and lipid metabolism by means the under regulation of FABP6 protein and FXR/RXR and LXR/RXR signaling pathway in ileum has been established for the first time in pigs. Taken together, our results provide a better understanding of the porcine response to Salmonella infection and the molecular mechanisms underlying Salmonella-host interactions., This research was supported by the National R&D Program of the Spanish Ministry of Science and Innovation (AGL2011-28904 and AGL2014-54089-R).

Background: The IL-1 family member IL-33 plays a critical role in type 2 innate immune responses to allergens and is an important mediator of allergic asthma. The mechanisms by which allergens provoke epithelial IL-33 secretion are still poorly understood.Objective: Based on previous findings indicating involvement of the NADPH oxidase dual oxidase 1 (DUOX1) in epithelial wound responses, we explored the potential involvement of DUOX1 in allergen-induced IL-33 secretion and potential alterations in airways of asthmatic patients.Methods: Cultured human or murine airway epithelial cells or mice were subjected to acute challenge with Alternaria alternata or house dust mite, and secretion of IL-33 and activation of subsequent type 2 responses were determined. The role of DUOX1 was explored by using small interfering RNA approaches and DUOX1-deficient mice. Cultured nasal epithelial cells from healthy subjects or asthmatic patients were evaluated for DUOX1 expression and allergen-induced responses.Results: In vitro or in vivo allergen challenge resulted in rapid airway epithelial IL-33 secretion, which depended critically on DUOX1-mediated activation of epithelial epidermal growth factor receptor and the protease calpain-2 through a redox-dependent mechanism involving cysteine oxidation within epidermal growth factor receptor and the tyrosine kinase Src. Primary nasal epithelial cells from patients with allergic asthma were found to express increased DUOX1 and IL-33 levels and demonstrated enhanced IL-33 secretion in response to allergen challenge compared with values seen in nasal epithelial cells from nonasthmatic subjects.Conclusion: Our findings implicate epithelial DUOX1 as a pivotal mediator of IL-33-dependent activation of innate airway type 2 immune responses to common airborne allergens and indicate that enhanced DUOX1 expression and IL-33 secretion might present important contributing features of allergic asthma.

Plants have developed a variety of mechanisms to cope with abiotic and biotic stresses. In a previous subcellular localization study of hydrogen peroxide-responsive proteins, two peptides with an unknown function (designated ARACIN1 and ARACIN2) have been identified. These peptides are structurally very similar but are transcriptionally differentially regulated during abiotic stresses during Botrytis cinerea infection or after benzothiadiazole and methyl jasmonate treatments. In Arabidopsis (Arabidopsis thaliana), these paralogous genes are positioned in tandem within a cluster of pathogen defense-related genes. Both ARACINs are small, cationic, and hydrophobic peptides, known characteristics for antimicrobial peptides. Their genes are expressed in peripheral cell layers prone to pathogen entry and are lineage specific to the Brassicaceae family. In vitro bioassays demonstrated that both ARACIN peptides have a direct antifungal effect against the agronomically and economically important necrotrophic fungi B. cinerea, Alternaria brassicicola, Fusarium graminearum, and Sclerotinia sclerotiorum and yeast (Saccharomyces cerevisiae). In addition, transgenic Arabidopsis plants that ectopically express ARACIN1 are protected better against infections with both B. cinerea and A. brassicicola. Therefore, we can conclude that both ARACINs act as antimicrobial peptides. ispartof: Plant Physiology vol:167 issue:3 pages:1017-1029 ispartof: location:United States status: published

Bioactive matrix fragments (matrikines) have been identified in a myriad of disorders, but their impact on the evolution of airway inflammation has not been demonstrated. We recently described a pathway where the matrikine and neutrophil chemoattractant proline–glycine–proline (PGP) could be degraded by the enzyme leukotriene A4 hydrolase (LTA4H). LTA4H classically functions in the generation of pro-inflammatory leukotriene B4, thus LTA4H exhibits opposing pro- and anti-inflammatory activities. The physiological significance of this secondary anti-inflammatory activity remains unknown. Here we show, using readily resolving pulmonary inflammation models, that loss of this secondary activity leads to more pronounced and sustained inflammation and illness owing to PGP accumulation. PGP elicits an exacerbated neutrophilic inflammation and protease imbalance that further degrades the extracellular matrix, generating fragments that perpetuate inflammation. This highlights a critical role for the secondary anti-inflammatory activity of LTA4H and thus has consequences for the generation of global LTA4H inhibitors currently being developed., Proteases degrade extracellular matrix during inflammation, releasing peptides that can recruit neutrophils. Here the authors show that degradation of such bioactive peptide by the enzyme leukotriene A4 hydrolase is critical to limit pulmonary inflammation during bacterial infection in mice.

T helper 1 driven immune responses facilitate host defence during viral infections. Because interleukin-18 (IL-18) mediates T helper 1 driven immune responses, and since mature IL-18 is up-regulated in human macrophages after influenza virus infection in vitro, it has been suggested that IL-18 plays an important role in the immune response to influenza. To determine the role of IL-18 in respiratory tract infection with influenza, IL-18 gene-deficient (IL-18(-/-)) and normal wildtype mice were intranasally inoculated with influenza A virus. Influenza resulted in an increase in constitutively expressed IL-18 in the lungs of wildtype mice. The clearance of influenza A was inhibited by IL-18, as indicated by reduced viral loads on day 8 and day 12 after infection in IL-18(-/-) mice. This enhanced viral clearance correlated with increased CD4(+) T-cell activation in the lungs as reflected by CD69 expression on the cell surface. Surprisingly, interferon-gamma (IFN-gamma) levels were similar in the lungs of IL-18(-/-) mice and wildtype mice. Intracellular IFN-gamma staining revealed similar expression levels in lung-derived natural killer cells, CD4(+) and CD8(+) T cells, indicating that IFN-gamma production is IL-18-independent during influenza virus infection. Tumour necrosis factor-alpha production by CD4(+) T cells was significantly lower in IL-18(-/-) mice than in wildtype mice. Our data indicate that endogenous IL-18 impairs viral clearance during influenza A infection.